Strep tag II Protein Purification Kit

1). Based on the estimated total amount of Strep-tag II tag protein in the sample, calculate the loading volume of the Streptactin-XT beads and add the required volume of beads to the gravity column.

 

2). After the beads to the bottom of the gravity column, equilibrate the beads with at least 3 column beads volumes of distilled water, and then equilibrate the beads with 5 column beads volumes of Binding / Equilibration Buffer (BE buffer for Streptactin-XT).

 

1). Adjust the sample to the composition of the binding buffer. For example, dilute the sample with Binding / Equilibration Buffer or buffer exchange using a desalting column.

 

2). Pass the sample through a 0.22 μm or a 0.45 μm filter and/or centrifuge it immediately before applying it to the column. If the sample is too viscous, to prevent it from clogging the column dilute it with Binding / Equilibration Buffer, increase lysis treatment (sonication, homogenization), or add DNase/RNase to reduce the size of nucleic acid fragments.

 

1). Add the clear sample containing the Strep-tag II fusion protein to the equilibrated gravity column, keep the sample in the column for at least 2 minutes to ensure fully binding of the sample to the beads, and collect the flow-through at a flow rate of 0.5-1 mL per minute (the flow-through valve can accurately control the flow rate). Then add the flow-through to the column again and repeat the previous operation. Repeated loading can improve binding efficiency. Collect the final flow through liquid for use as a control for subsequent testing.

 

 

1). Wash with 5 column beads volumes of Wash Buffer to remove non-specific proteins, repeat twice. Until the A280 of the ef?uent becomes stable at a flow rate of 1 mL/minute, 

2). The components of the washing flow through liquid were collected for use as negative controls for subsequent testing.

Important Note:

If high background (excessive non-specific binding) is observed in subsequent experiments, the duration and number of washing steps can be increased.

 

1). Elute with 3-5 column beads volumes of Elution Buffer (containing D-biotin), collect in batches, collect one tube per column volume, and detect separately. This ensures that all bound target proteins are eluted, while also obtaining high purity and concentration of the protein.

 

Note: Desthiobiotin cannot be used to elute this product.

In case of difficulty in protein elution, the concentration of D-biotin in the Elution Buffer and the number of elution cycles can be appropriately increased.

 

1). Wash with 5 column beads volumes of Wash Buffer

2). Regenerate the beads with 5 column volumes of Regeneration buffer for Streptactin-XT, Remove immediately.

3). Wash the beads with 8 column volumes of Wash Buffer, repeat twice.

4). After regeneration and wash, the beads is stored in an equal volume of Wash buffer at 2-8°C.